The biogenesis of adrenal chromaffin vesicles will be investigated by studying the incorporation of H3-leucine into the soluble and membrane-bound proteins of the vesicles. Isolated perfused bovine glands are pulse-labeled with H3-leucine and at various times afterward the adrenal medulla are homogenized and separated into subcellular fractions by differential and density gradient centrifugation. The soluble and membrane proteins of each of the fractions are analyzed for the total radioactivity and for patterns of radioactive pentides by SDS qel electrophoresis. Additionally specific incorporation of H3-leucine into dopamine-beta-hydroxylase and chromogranins is determined by immunoprecipitation. Analysis of the data will provide information on whether vesicle membranes are recycled. Additional studies are planned to study chromaffin vesicle formation in intact animals by labeling cat adrenal glands in situ by perfusion of H3-leucine through the adrenolumbar vein.